IL-13 cytotoxin has potent antitumor activity and synergizes with paclitaxel in a mouse model of oral squamous cell carcinoma Mitomu Kioi 1 , Takeshi Shimamura 1 , Hideyuki Nakashima 1 , Makoto Hirota 2 , Iwai Tohnai 2 , Syed R. Husain 1 and Raj K. Puri 1 * 1 Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 2 Department of Oral and Maxillofacial Surgery, Yokohama City University School of Medicine, Fukuura, Kanazwa-ku, Yokohama, Japan Interleukin-13 receptor-targeted cytotoxin (IL13-PE38) is highly cytotoxic to certain types of human cancers expressing abundant levels of IL-13Ra2 chain. Although IL13-PE38 is being tested in a Phase III clinical trial in brain tumors, the activity of IL13-PE38 alone or when combined with taxane, a chemotherapeutic drug for oral squamous cell carcinoma (OSCC), has not been investi- gated. Here, we show that approximately 40% of OSCCs (n 5 50) in a tissue array are strongly positive for IL-13Ra2, whereas nor- mal oral mucosa (n 5 10) expresses very low or undetectable lev- els evaluated by immunohistochemistry. IL13-PE38 was highly cy- totoxic to OSCC cell lines, but not cytotoxic to normal oral fibro- blasts. IL13-PE38 mediated a synergistic antitumor effect with paclitaxel in OSC-19 in vitro and in vivo in the orthotopic OSCC tongue tumor model. Real-time tumor growth was monitored by optical imaging using a Xenogen-IVIS imaging system. Treated ani- mals showed significant (p < 0.05) improvement in survival, which correlated with in vivo imaging of tumor response without evidence of visible toxicity. Gene transfer of IL-13Ra2 in oral cancer cells increased sensitivity of OSCC cell line to IL13-PE38 in vitro. Retro- virus-mediated gene-transfer of IL-13Ra2 in HSC-3 into tongue tumors in vivo dramatically enhanced the antitumor activity of IL13-PE38, providing complete elimination of established tumors and prolonging survival of these animals. These results indicate that IL13-PE38 in combination with paclitaxel acting via different mechanisms may be a potential treatment option for IL-13Ra2 expressing OSCC or for the treatment of non–IL-13Ra2 expressing OSCC combined with gene transfer of IL-13Ra2. Published 2008 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America. Key words: interleukin-13 receptor; squamous cell carcinoma; oral cancer; synergistic effect; gene transfer; retrovirus; paclitaxel; orthotopic animal model Cancer of the head and neck, including the oral cavity, is the sixth most common cancer in the United States. 1 The most com- mon type of oral cavity cancer is oral squamous cell carcinoma (OSCC). It accounts for 2% to 3% of all cancers in the United States, with approximately 34,000 new cases and 7,500 deaths per year. 1 Thus, OSCC remains a significant public health problem. In particular, SCC of the tongue is among the most common tumors of the head and neck. 2 Despite considerable advances in surgical techniques, irradiation delivery, and improvement in chemothera- peutic strategies, the 5-year survival rates for patients with OSCC in the past 30 years has remained 50% to 60%. 1 In particular, the 3–5 year survival rate of patients with advanced T3 and T4 stages of OSCC has remained poor (20%–30%). Given this dismal state, alternative therapies such as gene therapy and targeted therapy may give new hope for primary or adjuvant treatment for OSCC. Cytokines, growth factors, and their receptors play a pivotal role in the regulation or stimulation of cancer progression, neovas- cularization, immunosurveillance, and metastasis. 3–5 Interleukin-13 (IL-13) is one of the T cell-derived Type 2 cytokines that have been implicated in the above processes. 6 IL-13 binds to two receptor sub- units, IL-13Ra1 and IL-13Ra2, and stimulates downstream signal- ing cascades involved in cell proliferation and cytostatic effect or cell death of some neoplasic cells. IL-13Ra1 is a low affinity recep- tor but in association with IL-4Ra chain, it forms a high affinity complex; and IL-13 mediates signal transduction through this com- plex involving either JAK-STAT or PI3 kinase associated with cell proliferation, cell survival, and gene expression. 6 IL-13Ra2 chain binds IL-13 with high affinity and internalizes without the involve- ment of other chains. Although this receptor chain had been thought to be nonsignaling, it has recently been reported that IL-13Ra2 is involved in activation of the AP1 pathway leading to up-regulation of TGFb-1 production. 7 The expression of IL-13Ra2 has been shown to be elevated markedly in various malignant tumor cell lines and tissues derived from human malignant glioma, head and neck cancer, Kaposi’s sarcoma, ovarian cancer, and renal cell carcinoma. In contrast, normal cells or tissues derived from uninvolved locations of the tumor show very low or undetectable expression levels. 8–12 The extent of expression of IL-13Ra2 in head and neck carcinomas seems to be associated with the development of cancer. 9 The expression of this receptor has also been related to metastasis of breast cancer to lung. 13 Thus, targeting IL-13Ra2 is a reasonable strategy for treatment of certain cancers. To target IL-13R, IL-13 cytotoxin (IL13-PE38), which is com- posed of IL-13 and a mutated form of Pseudomonas exotoxin (PE) has been generated. 14 IL13-PE38 is highly cytotoxic to IL-13R- positive cancer cells in vitro and in vivo, and this cytotoxin has been investigated in several Phase I/II clinical trials in patients with glioblastoma. 8–12,14,15 In addition, a Phase III clinical trial was completed recently. 12 We previously demonstrated that not only IL-13Ra2-positive head and neck cancer cell lines but also IL-13Ra2-negative cell lines transfected with IL-13Ra2 become dramatically sensitive to IL13-PE38. 16 As OSCCs are fairly acces- sible tumors, a gene transfer approach can be easily applied to expand the therapeutic opportunity for targeting this cancer. In the current study, we examined the expression of IL-13R in OSCC cell lines and tumors in situ. We explored the antitumor effects of IL-13R targeted cytotoxin with or without gene transfer of IL-13Ra2 followed by IL13-PE38 therapy. In addition, we inves- tigated the potential of IL13-PE38 to enhance the effects of chemo- therapeutic agents for oral cancer therapy. Platinum drugs such as cisplatin and carboplatin, combined with Fluorouracil (5-FU), have been recognized as standard chemotherapy for OSCC. Recently, taxanes including paclitaxel and docetaxel, have been used in com- bination with platinum drugs to improve the prognosis. 17–19 As tax- anes can block cell replication, arrest cells in the G2 and M phase of the cell cycle, and stabilize cytoplasmic microtubules, a different mechanism of action than IL13-PE38, we explored whether IL13- *Correspondence to: Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Food and Drug Administration, Center for Biologics Evaluation and Research, NIH Building 29B, Room 2NN20, 29 Lincoln Drive, Bethesda, MD 20892, USA. Fax: 301-827-0449. E-mail: raj.puri@fda.hhs.gov Received 30 May 2008; Accepted after revision 24 September 2008 DOI 10.1002/ijc.24067 Published online 22 October 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 124, 1440–1448 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer